Synthesis, crystal structure, structural characterization and in vitro antimicrobial activities of 1-methyl-4-nitro-1H-imidazole

2.1 Synthesis and spectroscopic properties

Method A shows that 1-methyl-1H-imidazole can undergo nitration in a mixed acid (non fuming) 98% H₂SO₄ (non-fuming) and 65% HNO₃ at 5–65 °C bath temperature for 8 h. Instead of isomers or mixture of products we obtained only single regio-isomer namely 1-methyl-4-nitro-1H-imidazole in 20% yield as shown in Scheme 1. Temperature control and slow addition are important factors to obtain regio-specific nitration.

Method B shows that 1-methyl-1H-imidazole can undergo nitration in a mixture of urea nitrate/non-fuming conc. H₂SO₄ (98%). The method eliminates the use of liquid HNO₃ as the source for nitronium ion, and uses urea nitrate as an easy to handle source for nitration. Urea nitrate is known to convert deactivated aromatic compounds to the corresponding nitro derivatives with high regio-selectivity under mild conditions (Almog et al., 2006) but so far it has not been applied to make 1-methyl-4-nitro-1H-imidazole. The nitration process for the 1-methyl-1H-imidazole happens at low temperatures (−10 to +10 °C), so there is much less chance for non-regio-specific nitration. In addition the method gives better yield compared to Method A.

Spectroscopy such as, FTIR (Fig. SEI-1 and Table SEI-1), EIMS (Fig. SEI-2 and Scheme SEI-1), ¹H NMR (Fig. SEI-3), ¹³C NMR (Fig. SEI-4), and X-ray crystallography (Figs. 1 and 2) confirmed that the product is 1-methyl-4-nitro-1H-imidazole. FTIR spectrum data of compound (I) is compared (Table SEI-1) with the FTIR data (Acros Chemicals) of the starting material, which shows clear nitration of 1-methyl-1H-imidazole. EIMS spectrum (Fig. SEI-2) and clean fragmentation pattern of compound (I) are well explained (Scheme SEI-1). Important peaks appearing in the EIMS, at the m/z = 127, 111, 97, 81, and 54 arise due to the loss of single electron, loss of O, NO, NO₂, and loss of NO₂ & HCN respectively. The high intensity peak at m/z = 42 arises due to the loss of NO, CO and HCN from the molecular ion (Luijten and Thuijl, 1981).

¹H NMR of compound (I) showed 3 peaks at δ: 3.82, 7.42, and 7.76 ppm corresponding to CH₃, and two ArHs. We found that compound (I) shows ¹H NMR (400 MHz, CDCl₃) peaks at 7.76 (ArH, HC⚌N), 7.42 (ArH, HC⚌C), and 3.82 (N–CH₃) ppm instead of a previously reported (Katritzky et al., 2005a,b) ¹H NMR (300 MHz, CDCl₃) 7.78 (ArH), 4.72 (ArH), and 3.83 (N–CH₃) ppm. This might be a small correction of the printing error or typo but it is useful for future research. ¹³C NMR showed 4 peaks at δ: 34.6 ppm for CH₃, 120.1 for ArCH (HC⚌C), 136.6 for ArCH (HC⚌N), and 148.3 ppm for the ArC–NO₂ confirming the product compound (I).

2.2 X-ray crystallography

Single-crystal X-ray diffraction analysis was carried out to confirm the regio-specific nitration of the title compound (I). Crystal structure in our case is of very high importance to rule out the possibility of any other regio-isomer of compound (I). Crystal structure shows that compound (I) is composed of nitro and methyl groups attached to a planer imidazole ring. A detailed analysis of the molecule (Fig. 1) shows that all the bond lengths and angles are within expected ranges (Allen et al., 1987). The crystal structure is stabilized by the intermolecular hydrogen bonds (C–H⋯O–NO, 2.57 Å) and molecules are linked together to form sheets via C1---H1A⋯O2 running in a zig-zag fashion parallel to the a-axis (Fig. 2). Crystallographic data for structure determination and refinement of compound (I) are presented in Table 1. CCDC 875050 contains supplementary crystallographic data for compound (I). Data can be obtained free of charge via http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.

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Figure 1

ORTEP diagram of compound (I) with displacement ellipsoids drawn at 50% probability level.

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Figure 2

Crystal packing of compound (I); some hydrogen atoms were omitted for clarity.

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2.3 Structural properties

Properties of condensed phases are governed by the properties of the individual molecules due to intermolecular forces between molecules arising from electrostatic interactions (Buckingham, 1978). To probe into the structural properties of the 1-methyl-4-nitro-1H-imidazole or compound (I) we first optimized its molecular structure using Becke’s three-parameter density functional theory (DFT) in combination with Lee–Yang–Parr’s correlation functional (B3LYP) and employed 6-31G(d) basis sets (Lee et al., 1988; Becke, 1993) within the Wavefunction Spartan 08 (Yamada et al., 2011).

Fig. 3a shows the top view of the optimized structure showing corresponding atomic symbols. The side view (Fig. 3b) of the optimized molecular structure shows that 1-methyl-4-nitro-1H-imidazole is a planar molecule except the methyl group (N–CH₃) due to sp³ hybridization. This observation is consistent with its crystal structure (Fig. 1).

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Figure 3

Top view of the optimized structure (a), side view (b) and Mulliken charges (c) of the 1-methyl-4-nitro-1H-imidazole.

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Fig. 3c shows the Mulliken charges (Mulliken, 1955; Astrand et al., 1998) of different atoms in the optimized molecular structure. The nitro group shows the partial positive nitrogen (0.35) and partial negative oxygen atoms (−0.41 and −0.38) which make one end of the molecule partial negative (δ−) as shown in Fig. 3b. The nitrogen and carbon (N–CH₃) atoms showed partial negative charges (−0.38 and −0.33 respectively) while the hydrogen (N–CH₃) atoms showed partial positive charge (0.19 each), which makes the second corner of the molecule partially positive (δ+) as shown in Fig. 3b. The phenomenon of negative and positive polarizations created the driving force (Fig. 4) for crystallization and hydrogen bonding between the negatively charged oxygen atom of the nitro (NO₂) group and the positively charged hydrogen atom (Figs. 3b and 2) of the methyl group (N–CH₃). This observation also explains the special orientation of molecules in the crystal structure (Fig. 4). It can be observed from the crystal structure that only one oxygen atom of the nitro (NO₂) group participates in the H-bonding (Fig. 2), likely due to the more partial negative (−0.41 vs. −0.38) charge of one of the oxygen atoms bonded to NO₂.

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Figure 4

Proposed crystallization process for 1-methyl-4-nitro-1H-imidazole.

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2.4 Biological assay

For nitro-imidazoles two mechanisms of action based on previous studies have been reported (Van Der Wouden et al., 2000). Nitroimidazole enters the cell by passive diffusion, and is metabolized by a reduction step in which the drug behaves as the electron acceptor. For this reduction step several nitroreductases are present in Helicobacter pylori (bacteria). It has been suggested that an oxygen-insensitive NADPH nitroreductase encoded by the rdxA gene is the most important. Oxygen-insensitivity denotes in this case that the reduction of the nitroimidazole (Ar–NO₂) results in a nitrosoderivate (Ar–NO) by the simultaneous transfer of two electrons. This nitrosoderivate cannot be re-oxidized by molecular oxygen and the nitroreductase facilitating the two-electron transfer step is therefore called oxygen-insensitive. The nitrosoderivate leads to DNA damage and subsequent cell death of bacteria (Fig. 5, Mechanism #1).

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Figure 5

Anti-bacterial mechanism of nitroimidazole.

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The second mechanism explains that the other nitroreductases present in H. pylori reduce a nitroimidazole (R–NO₂) by a one-electron transfer step to a toxic free radical anion (R–NO₂)⁻ that can be metabolized in two ways (Fig. 5, Mechanism #2). First, the free-radical anion can be re-oxidized to the original compound (R–NO₂) by molecular oxygen with the production of superoxide (O₂⁻). Since molecular oxygen may reverse the reduction step these nitroreductases are called oxygen-sensitive. Theoretically, this process of reduction and re-oxidation is repeated endlessly and is called ‘futile cycling’. The superoxide produced during this ‘futile cycling’ is also toxic but can be eliminated (conversion to water) by superoxide dismutase (SOD) and catalase. The second way to metabolize the toxic free radical anion is another one electron transfer step to the more toxic nitrosoderivate that leads to DNA damage (inhibiting bacterial nucleic acid synthesis) and results in subsequent bacterial cell death of bacteria.

We have compared (as reference) the results with the standard drugs. For the antibacterial activity, we have used the gentamicin drug and for the antifungal, we have used the griseofulvin antifungal agent to draw meaningful comparison, the tables in the manuscript were generated considering all these parameters. This is a preliminary screening. We found encouraging results that the compound is active against pathogens. Antimicrobial activities show that the title compound is moderately active towards antibacterial and antifungal activities. Antimicrobial results for the title compound (I) and minimum inhibitory concentrations (MICs) for the bacterial and fungal isolates are reported in Tables 2 and 3 and the range is 180–300 μg/mL. Inhibition zones for Gram positive bacteria, Gram negative bacterial and fungal isolates were in the range of 16–18, 13–17 and 15–18 mm respectively. The maximum zone of inhibition was observed for Gram positive bacteria on Staphylococcus aureus AB 188 and for fungal isolate Candida tropicalis while 13 mm for Proteus mirabilis and Pseudomonas aeruginosa, 15 mm for Morganella morganii, Candida glabrata and Candida krusei, 16 mm for Micrococcus luteus ATCC 9341, S. aureus and Candida albicans ATCC 0383 and 17 mm for P. aeruginosa ATCC, Proteus vulgaris and C. albicans. In summary, antimicrobial results indicate that the title compound has the potential to kill some of the highly pathogenic bacterial and fungal species that have a critical role in a wide range of cutaneous, pyogenic and urinary tract infections (Gunay et al., 1999; Foroumadia et al., 2009).

4.1 Materials and methods

All chemicals and solvents were of reagent grade and purchased from Aldrich and used without further purification. Thin layer chromatography was performed on Merck silica gel 60F₂₅₄ plates and observed under UV. Melting point (uncorrected) was recorded in a glass capillary tube on Gallenkamp (sonyo) apparatus. FTIR spectra were recorded directly on Bruker FTIR spectrophotometer (Vector 22) in the range of 4000–400 cm⁻¹. EIMS spectra were recorded on JeolMS Route. ¹H NMR recorded on BrukerAvance 400 and ¹³C NMR recorded on BrukerAvance 300 spectrophotometers at 400 and 75 MHz respectively.

4.2 X-ray measurements

Single-crystal X-ray diffraction data were collected on Bruker Smart APEX II, CCD 4-K area detector diffractometer (Siemens, 1996). Data reduction was performed using SAINT program. The structure was solved by direct method (Altomare et al., 1993) and refined by full-matrix least squares on F² by using SHELXTL-PC package (Sheldrick, 1997). The figures were plotted with ORTEP program (Johnson, 1976).

4.3 Synthesis of compound (I)

Nitrating Mixture: Inside hood, nitrating mixture was prepared by mixing 4 mL of non-fuming H₂SO₄ (98%) and 3.8 mL of non-fuming HNO₃ (65%) in a round bottom flask with continuous stirring. HNO₃ was added slowly with the help of a dropping funnel into H₂SO₄ to maintain the temperature at 35–40 °C.

4.4 Synthesis methods for 1-methyl-4-nitro-1H-imidazole

Method A: Inside hood, to a round bottom flask fitted with a reflux condenser, was added nitrating mixture (7.8 mL) and the mixture was cooled down to 5–7 °C in an ice-salt cooled water-bath. To the cooled nitrating mixture drop-wise was added 1-methyl-1H-imidazole (1.019 g, 12.42 mmol) with continuous stirring. After 0.5 h of addition; bath temperature was allowed to increase temperature (60–65 °C) for 8 h. After this time, the reaction mixture was allowed to cool down to room temperature and the reaction mixture was added to a beaker containing purified water with stirrer. Mixture was transferred to a separatory funnel and methylene chloride (CH₂Cl₂) was added (50 mL × 3) to separate the organic phase. The organic phase was dried on anhydrous Na₂SO₄ and filtered. The CH₂Cl₂ was removed by rotary evaporator to afford the off-white solid, which is recrystallized with a small amount of CH₂Cl₂ to give the title compound (I) in 20% yield.

Method B: Inside hood, to a 200 mL round bottom flask precooled to −10 °C, freshly prepared dry solid 2.5 g (20.31 mmol) urea nitrate (Shead, 1967) was added in small portions to the liquid 1-methyl-1H-imidazole (1.019 g, 12.42 mmol, density = 1.03 g/cm³) and H₂SO₄ (15 mL). After the complete addition, the temperature was slowly allowed to increase up to 10 °C and maintained for 6 h. The mixture was then poured into a beaker containing crushed ice made from distilled water. After the complete melt down, the acidic solution was neutralized by aqueous Na₂CO₃ solution. The organic phase was separated with EtOAc and filtered through a small plug of dry silica and Na₂SO₄ in a Buchner funnel. EtOAc was used to wash silica and Na₂SO₄, and the solvent was removed to obtain pure product. This process afforded the off-white title compound (I) in 45% yield.

High quality single crystals suitable for X-ray crystallography were grown in a mixture of solvents chloroform and hexane (1:1) by slow evaporation at room temperature; mp: 133–134 °C. FTIR (KBr, cm⁻¹): v 3115 (⚌C–H arom. str.), 2925 (C–H aliphatic str.), 1424 (C⚌C ring str.), 1525, 1321 (NO₂ Asym./Sym. str.), 982 (C–N). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 3.82 (s, 3H, CH₃), 7.42 (s, 1H, ArH, HC⚌C), 7.76 (d, J = 1.0 Hz, 1H, ArH, HC⚌N). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 34.6 (CH₃), 120.1 (ArCH, HC⚌C), 136.6 (ArCH, HC⚌N), 148.3 (ArC–NO₂). EIMS m/z (%): 127 (M⁺, 100), 111 (10.6), 97 (5.4), 81 (12.4), 69 (12.7), 54 (11.1), 42 (78.5). Anal. Calcd. for C₄H₅N₃O₂: C, 37.80; H, 3.97; N, 33.06. Found C, 38.01; H, 3.93; N, 32.96.

4.5 Antimicrobial assays

Antibacterial activity: In the present study, synthetic compound (I) was monitored for antibacterial activities against Gram positive and Gram negative bacteria. All the bacterial isolates were checked for purity and maintained on nutrient agar at 4 °C in refrigerator prior to use. Antibacterial activity of the title compound (I) against pathogenic bacteria was determined using the agar-well method. Autoclaved Muller Hinton broth (Oxoid, Basingstoke–UK) was used to refresh the bacterial culture, later wells were punched into Muller Hinton Agar and 10 microlitres of culture were poured into the wells (Perez et al., 1990). All plates were incubated at 28 ± 2 °C for 24–48 h and after incubation the diameter of the inhibition zone was measured. Gentamicin antibiotic was used as a control agent in testing.

Antifungal activity: Test organisms for this study were members of the 6 saprophytic fungi Penicillium sp., Aspergillus flavus, Aspergillus niger, Fusarium sp., Rhizopus, Helminthosporium and Neurospora, 5 dermatophytic Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans and 6 yeasts including C. albicans, C. albicans ATCC 0383, Saccharomyces cerevisiae, C. glabrata, C. tropicalis, C. krusei. All the fungal isolates were checked for purity and maintained on Sabourd Dextrose Agar (SDA) (Oxoid, Basingstoke–UK) at 4 °C in the refrigerator until required for use. Antifungal activity of the title compound against human, environmental and phyto-pathogenic fungi was determined by using the agar-well method. Autoclaved distilled water was used for the preparation of fungal spore suspension and transferred aseptically into each SDA plates (Cappuccino and Sherman, 2007). All plates were incubated at 28 ± 2 °C for 24–48 h and after incubation, the diameter of the zone of inhibition was measured. Griseofulvin antifungal agent was used as a control.

4.7 Minimum inhibitory concentration (MIC)

Minimum inhibitory concentration (MIC) of the synthetic compound (I) was determined by the Micro broth dilution method using 96-well microtitre plate (Samie et al., 2005). Stock solution of 10 mg/mL was prepared in chloroform, two fold serial dilutions were made in 100 μL broth (Oxoid, Basingstoke–UK) and subsequently 10 μL of 2 h old refresh culture matched with 0.5 Mac Farland index was inoculated into each well. One well served as antibiotic and antifungal control while others served as culture control to check MIC. Microtitre plate was incubated for 24 h at 37 °C. The MIC showed no visible growth.